This project will develop new tools to facilitate high throughput, genome-wide mutagenesis in mouse embryonic Stem (ES) cells. Functional analysis of the mouse genome is a critical component of functional genomics, providing information relevant to human gene function and disease states. Gene trap mutagenesis in ES cells is a proven technology for generating sequence-tagged insertional mutations in mice. We propose to develop new gene trap vectors, incorporating a novel integrase-mediated recombination system. This vector will be used to generate a library of ES clones containing insertions within a wide range of genes across the genome. This library will be screened for expression via in vitro expression assays, using differentiation protocols already developed in the group. Each insertion will also have a RACE sequence tag associated with it and all information will be incorporated into an online database for availability to the community. Interesting insertions can be used to study mutant phenotype after germ line transmission, or can be used as sites of further gene modification or addition, by means of the integrase system. A complementary strategy of mutagenesis in ES cells will also be developed, focusing on in vitro recessive phenotypic screening, combining chromosome-specific loss of heterozygosity with chemical mutagenesis of ES cells. Both strategies will be developed first in existing 129 ES cells, but will be transferred to newly derived C57BL/6 cell lines, as soon as they are validated, to provide mutations on the B6 'gold standard' background.